Hepatitis B virus (HBV) is a member of a group of small DNA-containing viruses that cause persistent noncytopathic infections of the liver. HBV infection in humans can cause severe jaundice, liver degeneration and death. HBV enters predominantly by the parenteral route, has a characteristic incubation period of 60 to 160 days, and may persist in the blood for years in chronic carriers. HBV is of great medical importance because it is one of the most common causes of chronic liver disease, such as hepatocellular carcinoma, in humans. Infected hepatocytes continually secrete viral particles that accumulate to high levels in the blood. Moreover, it is estimated that about 6 to 7% of the human population is infected, with the level of infection being as high as 20% of the population in certain regions of Southeast Asia and sub-Sahara Africa.
Several tests have been employed to detect the presence of HBV constituents in serum and other body fluids. These tests are primarily immunological in principle and depend on the presence of antibodies produced in humans or animals to detect specific viral proteins such as the hepatitis B surface antigen (HBsAg), hepatitis B core (nucleocapsid) antigen (HBcAg) or hepatitis B “E” antigen (HBeAg). However, there are increasing concerns about the contribution of variant HBsAgs relative to the production of false negatives in serological HBsAg diagnosis or blood screening assays.
In particular, HBV, due to its mode of replication by reverse transcription of its pre-genomic RNA, has a high rate of mutation relative to other DNA viruses. Amino acid substitutions have been described in all HBV DNA-encoded viral proteins such as polymerase, HBcAg and HBsAg. The group-specific “a” determinant region of HBV (amino acids 124-147, numbered relative to the S portion of HBsAg) has attracted the most attention, because mutations in this region have been found in 10-20% of vaccine escapees and have resulted in the misdiagnoses of variant HBVs, even using the most current serological assays on the market. Thus, there is a need for the development of reliable diagnostic tests to detect HBV in viremic samples, in order to prevent transmission of the virus through blood and plasma derivatives or by close personal contact.
Rabbit-rabbit and rabbit-mouse hybridomas have been used in an attempt to generate monoclonal antibodies with increased immunoreactivity. See, e.g., U.S. Pat. Nos. 4,977,081; 4,859,595; 5,472,868; 5,675,063; Spieker-Polet et al., Proc. Natl. Acad. Sci. USA (1995) 92:9348-9352. Rabbit monoclonal antibodies are desirable for several reasons. First, rabbits may recognize antigens and epitopes that are not immunogenic in mice or rats, the two species from which monoclonal antibodies are usually generated. Additionally, rabbit antibodies are generally of high affinity. U.S. Pat. No. 4,859,595 describes the production of rabbit monoclonal antibodies to HBsAg using rabbit-rabbit fusions.
However, there remains a need for improved immunoassays using monoclonal antibodies with broader immunoreactivity against the various HBsAg mutants. The wide-spread availability of reagents for use in an accurate and efficient assay for HBV infection would be highly desirable.